We describe the first freshwater members of the class Actinobacteria that have been isolated. Nine ultramicro-size (<0.1 m 3 ) strains were isolated from five freshwater habitats in Europe and Asia. These habitats represent a broad spectrum of ecosystems, ranging from deep oligotrophic lakes to shallow hypertrophic lakes. Even when the isolated strains were grown in very rich media, the cell size was <0.1 m 3 and was indistinguishable from the cell sizes of bacteria belonging to the smaller size classes of natural lake bacterioplankton. Hybridization of the isolates with oligonucleotide probes and phylogenetic analysis of the 16S rRNA gene sequences of the isolated strains revealed that they are affiliated with the class Actinobacteria and the family Microbacteriaceae. The previously described species with the highest levels of sequence similarity are Clavibacter michiganensis and Rathayibacter tritici, two phytopathogens of terrestrial plants. The 16S rRNA gene sequences of the nine isolates examined are more closely related to cloned sequences from uncultured freshwater bacteria than to the sequences of any previously isolated bacteria. The nine ultramicrobacteria isolated form, together with several uncultured bacteria, a diverse phylogenetic cluster (Luna cluster) consisting exclusively of freshwater bacteria. Isolates obtained from lakes that are ecologically different and geographically separated by great distances possess identical 16S rRNA gene sequences but have clearly different ecophysiological and phenotypic traits. Predator-prey experiments demonstrated that at least one of the ultramicro-size isolates is protected against predation by the bacterivorous nanoflagellate Ochromonas sp. strain DS.The majority of the bacterial cells present in the bacterioplankton of marine and freshwater habitats are small (cell length, Ͻ1.5 m; volume, Ͻ0.3 m 3 ). Even very small bacteria, which are termed ultramicrobacteria (Ͻ0.1 m 3 ), are always present, and they frequently represent the numerically predominant fraction of typical marine and freshwater bacterioplankton. In contrast, in culture the cells of almost all bacteria isolated from bacterioplankton are much larger (cell length, Ͼ1.5 m; volume, Ͼ0.3 m 3 ) than most planktonic bacteria. In the case of several normally large-cell-size bacterial species, it has been shown that under strong starvation conditions the cell size decreases to dimensions typical of the majority of planktonic bacteria (23,26,27,31). There are only a few bacterial strains that have been isolated from marine and soil habitats whose cells are in the ultramicro size range (volume, Ͻ0.1 m 3 ) during growth under lab conditions (4,6,19,20,29,32,33,38). The most intensively investigated ultramicrobacterium is Sphingopyxis alaskensis (formerly Sphingomonas alsakensis [10,37]), a marine bacterium belonging to the class Alphaproteobacteria. Strains of this species have been isolated from Resurrection Bay, Alaska, from the North Sea, and from coastal waters near Japan (6,32,33). Recently, Rappé et...

No abstract

We isolated 28 strains of 'Spumella-like' flagellates from different freshwater and soil habitats in Austria, People's Republic of China, Nepal, New Zealand, Uganda, Kenya, Tanzania and Hawaii by use of a modified filtration-acclimatization method. 'Spumella-like' flagellates were found in all of the samples and were often among the dominant bacterivorous flagellates in the respective environments. The small subunit ribosomal RNA (SSU rRNA) gene sequence of the isolates was determined and aligned with previously published sequences of members belonging to the Chrysophyceae sensu stricto. Phylogenetic analysis of the 28 new sequences confirmed their position within the Chrysophyceae sensu stricto and positioned them within different clades. Most of the sequences grouped within clade C and formed several subclusters separated from each other by green taxa including flagellates belonging to Ochromonas, Dinobryon, Poterioochromonas and others. All soil isolates clustered together (subcluster C1) with the soil strain Spumella elongata and the undescribed soil strain 'Spumella danica'. Aquatic isolates were affiliated with at least two branches (C2 and C3). Sequence similarity to the closest related member of the Chrysophyceae ranged between 92% and 99.6%, sequence divergence among the 'Spumella-like' flagellates was as high as 10%. We conclude that (i) the 'Spumella-like' flagellates are a diverse group both in terms of sequence dissimilarity between isolates and in terms of the number of genotypes, (ii) Spumella and Ochromonas are polyphyletic, and (iii) based on the SSU rRNA gene no biogeographical restriction of certain branches could be observed even though different ecotypes may be represented by the same genotype.

Ultramicrobacteria (cell volume < 0.1 m 3 ) are the numerically dominant organisms in the plankton of marine and freshwater habitats. Flagellates and other protists are assumed to be the most important predators of these ultramicrobacteria as well as of larger planktonic bacteria. However, due to controversial observations conducted previously, it is not clear as to whether fractions of the ultramicrobacteria are resistant to flagellate predation. Furthermore, it is not known if closely related bacteria vary significantly in their sensitivity to flagellate predation. We investigated the sensitivity of ultramicrobacteria affiliated with the cosmopolitan Polynucleobacter cluster to grazing by Spumella-like nanoflagellates. Laboratory grazing experiments with four closely related (>99.6% 16S rRNA gene sequence similarity) bacteria and three closely related (100% 18S rRNA gene sequence similarity) flagellates were performed. In comparison to larger bacteria, predation on the ultramicrobacterial Polynucleobacter strains was weak, and the growth of the predating flagellates was slow. Specific clearance rates ranged between 0.14 ؋ 10 5 and 2.8 ؋ 10 5 units of predator size h ؊1 . Feeding rates strongly depended on the flagellate and bacterial strain (P < 0.001). Grazing mortality rates of the three flagellate strains investigated varied for the same prey strain by up to almost fourfold. We conclude that (i) ultramicrobacteria affiliated with the Polynucleobacter cluster are not protected from grazing, (ii) strain-specific variations in grazing sensitivity even between closely related bacteria are high, and (iii) strain-specific differences in predator-prey interaction could be an important factor in the evolution and maintenance of microbial microdiversity.It has been postulated that size-selective grazing of protists tends to select the small bacteria that are found to dominate in most natural waters (6, 23). Even though the ingestion of virus particles (15) and high-molecular-weight polysaccharides (30) has been demonstrated, it is undoubted that smaller bacteria experience lower grazing pressure than larger bacteria (1,13,14,23,29). For the smallest bacteria, i.e., ultramicrobacteria (Ͻ0.1 m 3 ), whether complete grazing protection is due to their small size or insufficient capturing devices of their predators is still in discussion. For larger bacterivores, such as cladocerans and some ciliate species, it is well accepted that the smallest bacteria escape grazing. In contrast, nanoflagellates are generally assumed to also graze on small bacteria (14, 25). Even flagellate species that usually prey on larger bacteria are able to ingest the smallest particles, i.e., virus-like particles (15), high-molecular-weight polysaccharides (30), and artificial particles less than 0.4 m in diameter (unpublished data). On the other hand, grazing protection against Ochromonas has been demonstrated for an ultramicrobacterial strain (19).It is thus still controversial whether ultramicrobacteria are subject to nanoflagellate predatio...

The combined effect of temperature and food concentration on the growth rate, cell volume, and production of the freshwater ciliate Urotricha farcta was investigated in laboratory batch cultures. Experimental temperatures ranged from 9 to 25ЊC and food levels ranged from 0.1 to 4.4 g C ml Ϫ1. The ciliates were fed the small cryptophyte Cryptomonas sp. The combined effect of temperature and food on growth and cell volume resulted in negative production rates at high temperatures and low to moderate food supply. Three main changes were observed in the shape of the numerical response (growth vs. food concentration) of U. farcta with temperature: change in the threshold level, where population net growth rates are zero; change in the initial slope of the numerical response curve; and change in maximum growth rate ( max ). The threshold food concentration and max were shifted up at the highest temperatures. The threshold level was also higher at the lowest experimental temperature. The initial slope of the numerical response curve was several-fold lower at both high and low temperatures. The analysis suggests that temperature altered the numerical response so that the species shifted from being adapted to low food concentrations at moderate temperatures to requiring, and potentially thriving at, high food concentrations at the temperature extremes. These findings support and extend conclusions previously obtained for metazooplankton and indicate that changes as small as 3ЊC could alter the role of protozoa in planktonic food webs.